Erythropoietin (EPO) is a glycoprotein hormone which controls the formation of erythrocytes from precursor cells in the bone marrow (erythropoiesis). EPO binds here to its receptor (EPO-R), which is expressed in all haemopoietic cells.
In adults, erythropoietin is formed principally in the kidney, more precisely in the endothelial cells of the peritubular capillaries. Relatively small amounts are also synthesised in the liver cells (hepatocytes).
The principal action of EPO thus consists in increasing the number of red blood corpuscles in the blood, which results in increased oxygen uptake.
In recent years, diverse authors have reported that EPO also exerts a non-haemopoietic action and EPO-R is correspondingly also expressed by certain non-haemopoietic cells. Thus, stimulation by EPO of nerve cells, neuronal cells of the brain and endothelial cells is reported, in some cases together with direct expression of the haemopoietic EPO receptor. In other cases, the presence of a further, non-haemopoietic receptor is prognosticated, but this has not yet been proven.
Increasing importance is being accorded, in particular, to the non-haemopoietic action, which has not been known for very long, of erythropoietin (EPO) in connection, for example, with the stimulated formation and regeneration of endothelial and tissue cells, such as connective tissue, muscle tissue, epithelial tissue and nerve tissue.
Thus, WO 2004/001023 describes, inter alia, the use of EPO and TPO for stimulating neovascularisation and tissue regeneration and improving wound healing, for example after operations or injuries.
WO 2005/063965 teaches the use of EPO for the targeted, structurally controlled regeneration of traumatised tissue, in which not only is endothelial cell growth stimulated, but parenchymal regeneration and the formation of wall structures are also promoted, meaning that coordinated three-dimensional growth occurs for the construction of a functioning tissue, organ or parts thereof.
Haroon et al. (American J. Pathol. 2003, 163, 993) discuss the new role of EPO in the context of wound healing processes induced by fibrin.
In a review article, Brines and Cerami (Kidney International, 2006) discuss the role of EPO in the protection of tissue.
Erythropoietin, and EPO derivatives or also EPO mimetics, thus appear to be highly suitable on systemic use for specifically initiating and controlling neoformation and regeneration of the affected tissue in the case of injuries to the skin, the mucous membrane, in the case of open skin and flesh wounds or also in the case of skin irritation caused by burns or scalds, and is ultimately able to promote and accelerate healing.
WO 2005/070450 and further papers by the inventors in question describe the use of EPO in the regeneration of vessels and tissue with a weekly dose of less than 90 IU/kg of body weight (=BW), including for the area of wound care. Although possible topical application is theoretically mentioned here, it has nevertheless been found that systemic application is preferred.
It is therefore postulated to administer EPO in the case of systemic application in a sub-polycythemic weekly dose of less than 90 IU (international units)/kg of body weight (BW) instead of 150-300 IU/kg of BW, as was hitherto usual for the known EPO applications. The aim is thus to achieve less stimulation of blood formation in the bone marrow region, but, according to more recent teaching, as outlined, to enable activation of endothelial cell progenitors in the blood region. Activation of the endothelial cell precursor cells in the blood, but also in the tissue, and the development of endothelial cells, which form the innermost cell layer of the blood vessels, have been connected with an improvement in vascularisation, and it is assumed that tissue regeneration is also facilitated thereby. In the meantime, this has been confirmed in clinical trials in the case of burn wounds.
Although some of the said papers mention topical application of EPO for the regeneration of tissue, systemic application of the active compound is, however, placed clearly in the foreground since, on the basis of the results, the non-haemopoietic EPO effect found can be attributed, in the opinion of more recent papers, primarily to the newly discovered stimulation of corresponding endothelial, vascular or CD31-positive precursor cells, which circulate principally with the blood stream, and only secondarily to the growth of parenchymal tissue structures stimulated thereby.
However, the use of EPO in a systemic application for tissue protection or tissue regeneration is associated with significant risks owing to the side-effect potential in relation to the haemopoietic effects.
On topical application of EPO, according to teaching opinion to date, merely the inadequate distribution and reachability of the said systemically occurring cells or precursor cells by EPO means that only an unsatisfactory effect, or none at all, on tissue regeneration would be observed.
In the case of regeneration of tissues after, for example, burn trauma or scalding or also in the case of ischaemic wounds, it is necessary to achieve rapid defect closure. This can only occur if the formation of the parenchymal components of the skin is also stimulated as quickly as possible. The time-shifted stimulation of one component (CD31), in order then to facilitate the formation of another component (parenchyma), corresponds to the teaching of the authors of WO 2005/070450 and further publications.
Although the formation of a vascular network can have an indirect supporting action, it does not as such represent an end result, since the parenchyma is missing and under certain circumstances can only be formed secondarily and thus with a time delay.
The formation of vascular cells must thus be coordinated simultaneously with localised tissue formation. In this case, in accordance with conventional teaching, a dilemma occurs, since doses administered subcutaneously or intravenously target endothelial cell progenitors owing to a systemic distribution principle, and sub-polycythemic doses must additionally be administered in order to limit side effects. In this case, the administration of EPO can thus only be combined with difficulty with localised pharmacokinetics limited locally to the trauma region of the skin.
In accordance with conventional teaching, conceptional stimulation pathways must therefore be selected, where either sub-polycythemic doses must be administered or repeated injections or also changes in the half-value period of the parent substance are necessary.
In each case, however, EPO will arrive at the actual site of need via the bloodstream systemically distributed in the body and even further diluted.
Furthermore, processes occur in the case of wounds which make topical application of EPO more difficult: wound healing of, for example, injured skin or mucous membrane usually proceeds in three phases: the inflammation phase, the proliferation phase and the restoration/remodelling phase. In the case of a fresh wound or skin injury to be cared for, inflammatory processes occur within the first 24 hours, which include, in particular, the immigration of diverse inflammation factors (such as, for example, fibronectin) and cells of various types, such as, for example, monocytes, phagocytes, polymorphic cells and macrophages, and ultimately result in the formation of a fibrin matrix and vascular endothelial cells. The wound secretion formed in the process comprises, inter alia, a series of proteolytic enzymes as well as bacteria which have entered the wound and comprise substances which act in this respect.
The proteolytic enzymes, some of which are highly active, are the reason why protein- or peptide-containing medicaments, such as EPO, which have been applied to the wound and promote wound healing are often of little or no efficacy, since the protein or polypeptide in question is, owing to its chemical and biological nature, deactivated, cleaved and degraded by said enzymes before it is able to develop adequate pharmacological efficacy. The problem is additionally exacerbated by infection of the wound with bacteria or the ingress of cell debris.
Not least for this reason, pharmaceutical proteins are generally applied systemically, which enables their half-value period to be significantly extended and also enables them to be transported more quickly to the sites in the body at which they are to develop their therapeutic efficacy. However, the doses of the protein-containing active compound must be sufficiently high in this application method in order to achieve the desired therapeutic effect, which often inevitably results in undesired side reactions.
In the case of the therapeutic treatment of skin injuries, systemic application of an active compound moreover in principle appears less appropriate, since the healing action of the medicament is actually only necessary locally. There is thus a general problem if protein-containing active compounds are to be employed for the treatment of skin injuries and open flesh and skin wounds.
The use of proteins or polypeptides in topical form, such as, for example, EPO, which, even in plasma, has a half-value period of only 48 hours, for the treatment of skin injuries of this type, as may occur in the case of violent mechanical trauma and irritation and in the case of burns and scalds, would, however, be very desirable in spite of the known difficulties.
The object is thus to provide EPO or its bioequivalent derivatives, fragments, mimetics and the like for skin wound healing in the form of a topical application, without on the one hand, as just described, dramatic losses of action occurring due to proteolysis of EPO due to enzymatic or other processes in the wound, but on the other hand, as likewise explained in detail above, possibly facilitating stimulation of cells or precursor cells which are capable of wound healing in the immediate wound region and if possible also in deeper tissue layers in the vicinity of the wound by topically applied EPO, which ultimately should result in faster wound closure and makes topical application of EPO appear sensible for the first time and provides it with a significant advantage over systemic administration.
A further aim of the invention is to create a form of administration which does not have to take into account the risks and restrictions of a systemic, in particular sub-polycythemic dosage of EPO, and at the same time can act specifically on the actual tissue-relevant cells without at the same time excluding the localised adult endothelial cells.